Cyclin-dependent Kinase 1 In Litopenaeus Vannamei:Molecular Characterization And Its Response To Cold Stress

The Pacific white shrimp(Litopenaeus vannamei)is one of the major aquacultural shrimp species in worldwide.In recent years,global warming has caused climate disasters frequent,which has dramatically influenced the development of aquaculture industry.Temperature is one of the important environmental factors affecting the cultivation of L.vannamei and has a significant impact on its growth,development,neuroendocrine,antioxidative stress system,immune system,etc.and even leads to the death of shrimp,causing huge economic losses.Under the basis of the low temperature transcriptome of L.vannamei muscle in the early laboratory,the key response gene Cdk1 of L.vannamei under low temperature stress was mined by bioinformation analysis,the full length of Lv-Cdk1 cDNA was cloned by RACE,its distribution in cells was determined by subcellular localization,r Lv Cdk1 was expressed by prokaryotic expression technology,its distribution in the hepatopancreas was explored by in situ hybridization.The tissue distribution of Lv-Cdk1 mRNA in shrimp and expression changes of Lv-Cdk1 mRNA under different environmental factors was explored by qPCR.After injecting dsRNA to silence Lv-Cdk1,the function of Lv-Cdk1 was explored.The main contents are as follows:1.Molecular characteristics of Lv-Cdk1Based on the low temperature transcriptome data,the key response gene Cdk1 was screened out,and the full cDNA length of the Chinese shrimp was cloned by RACE based on the reference sequence in the whole genome data of L.vannamei(NCBI,XM＿027370494.1).Lv-Cdk1 cDNA has a total length of 1467 bp,an ORF of 900 bp,a 5’-UTR of 42 bp,a 3’-UTR of 525 bp,encoding 299 amino acids,a S＿TKc domain at positions 4-287,conserved phosphorylation of Thr14 and Tyr15 and dephosphorylation sites of Thr161,and conserved elements PSTAIRE,DFG,and GxGxxGx V.The theoretical isoelectric point of Cdk1 is 8.46,without any signal peptides,present in the membrane,with the typical bilobe structure of Cdks kinase,and the predicted relative molecular weight is 34.7 kDa.Evolutionary relationship analysis showed that Lv-Cdk1 was most closely related to the Portunus trituberculatus.2.Distribution characteristics of Lv-Cdk1 and transcription level of Lv-Cdk1 under different environmental stressesSubcellular localization showed that Lv-Cdk1 was present in the nucleus and cytoplasm.The relative molecular mass of r Lv Cdk1 expressed by prokaryotes was 34.5 kDa,which was consistent with the predicted relative molecular mass of Cdk1.The mRNA of Lv-Cdk1 was expressed in tissues of gill,heart,hepatopancreas,intestine,muscle,stomach,brain,nerve,among which the relative expression was highest in the hepatopancreas.In situ hybridization further confirmed that Lv-Cdk1 was expressed in large quantities in the hepatopancreas,mainly in the distal region.The relative expression of Lv-Cdk1 mRNA was significantly upregulated under the physical challenges of low temperature,high temperature,low pH and low salt,and the relative expression of Lv-Cdk1 mRNA was downregulated under LPS and Poly(I:C)biological stress.3.The function of Lv-Cdk1After injecting dsRNA silencing the Cdk1 gene,the survival rate of L.vannamei was only50 % after 12 h in the cold stress experimental group,while the survival rate of shrimp in the blank control and negative control experimental group was higher than 90 %.After Lv-Cdk1 was silenced,the relative expression of apoptotic genes P53,Bcl-2,Cytochrome c,and Caspase-3 mRNA was significantly upregulated at low temperature,and the relative expression of immune genes Relish and Crustin mRNA also changed dramatically.In summary,Lv-Cdk1 has a high degree of similarity with crustaceans and is mainly expressed in the hepatopancreas.Lv-Cdk1 mRNA expression changed significantly after environmental stress.After Lv-Cdk1 was disturbed,the survival rate of L.vannamei in low temperature was significantly reduced,and the relative expression of mRNA of apoptotic genes and immune genes changed significantly,indicating that Lv-Cdk1 plays an important role in the response of cold stress in L.vannamei.The results in the thesis provide data for the depth exploration of L.vannamei response to cold stress,and provide theoretical guidance for molecular breeding of low temperature tolerant shrimp.